Citation of any reference in this section or any section hereof is not to be construed as an admission that such reference is available as prior art to this invention.
Controlled expression of transgenes or of target endogenous genes is essential for success of many genetic therapies. Constitutive expression of transgenes has often resulted in down-regulation of effector systems and/or cellular toxicity in animal studies (see, e.g., Efrat et al., 1995, Proc. Natl. Acad. Sci. USA 92, 3576-3580).
The expression of endogenous eukaryotic genes is often regulated by, for example, metabolic, hormonal, or environmental signals. In order to mimic natural physiological expression patterns with transgenes, and to minimize interactions with human gene regulation signals, binary promoter/transactivator configurations of heterologous origin which respond to heterologous stimuli have been developed in recent years. However, many exogenous stimuli which modulate these artificial mammalian regulons have proven to be incompatible with human therapeutic use due to cytotoxicity or undesired side effects (Baim et al., 1991, Proc. Natl. Acad. Sci. USA 88, 5072-5076; Braselmann et al., 1993, Proc. Natl. Acad. Sci. USA 90, 1657-1661; No D. et al., 1996, Proc. Natl. Acad. Sci. USA 93, 3346-3351; Rivera et al., 1996, Nat. Medicine 2, 1028-1032; Suhr et al., 1998, Proc. Natl. Acad. Sci. USA 95, 7999-8004; Wang et al., 1994, Proc. Natl. Acad. Sci. USA 91, 8180-8184; Fussenegger et al., 2000. Nat. Biotech. 18, 1203-1208).
Two systems have had some success for regulation of expression of transgenes in mammalian cells. In particular, the streptogramin-regulated mammalian expression system and the tetracycline-regulated mammalian expression system have avoided some of the problems associated with previous efforts. Streptogramin-regulated systems and tetracycline-regulated systems are described in U.S. Pat. Nos. 5,888,981; 5,866,755; 5,789,156; 5,654,168; and 5,650,298; PCT application no. WO 00/65080, to name just a few examples. However, the tetracycline-regulated system can fail to achieve the desired regulatory effects like low leakyness under repressed conditions or maximum expression in the induced state.
Moreover, gene therapy strategies often require independent control of multiple different transgenes or sets of transgenes which are cotranscribed in a multicistronic configuration. For example, many tissue expansion and ex vivo gene therapy scenarios require a two-step process beginning with expression of growth-promoting genes to enable expansion of grafted tissues in culture, followed by induction of growth suppressors to prevent tumorigenic behavior of treated cells after reimplantation. Sustained proliferation control is also required for stem cell-based technologies currently evaluated for eventual cell and tissue replacement therapy, since stem cells are tumorigenic (Rossant et al., 1999, Nat. Biotechnol. 17, 23-24; Solter et al., 1999, Science 283, 1468-1470). A second, independent gene regulation system could be used in such cells for pharmacologic control of one or several secreted therapeutic proteins, such as insulin, to enable titration of circulating proteins into the therapeutic range or adapt expression to optimal daily dosing regimes.
There is, therefore, a need for new mammalian gene regulation systems that employ modern, therapeutically proven antibiotics as controlling agents, and which can be used in combination with the tetracycline and/or streptogramin regulation systems, with minimal interaction between either tetracycline control or streptogramin control, or both, and the new control modality. However, dual-regulated expression technology is not sufficient to enable construction of complex artificial regulatory networks and cascades required for more sophisticated multigene interventions in next-generation human therapies and biopharmaceutical manufacturing. A novel mammalian gene regulation system that is independent of the streptogramin and tetracycline-regulated systems is needed to enable the regulation of such complex networks and cascades.